COFACTOR ROLE IN BETA-SHEET PROTEIN FOLDING

描述：(改编自申请者的摘要)辅因可能很重要 决定因素，作为限制构象搜索的成核点， 辅因子结合蛋白的折叠率。迄今为止，折叠动力学 对具有b-折叠结构的蛋白质的研究还没有 对于螺旋蛋白质来说。这项研究计划旨在探讨 三种蛋白质折叠中的两种无机辅因子和一种有机辅因子 主要是B-Sheet结构。目标蛋白是天青素，一种b桶蛋白。 含有铜离子辅因子黄毒素，一种a/b双链的蛋白质 配位有机黄素单核苷酸(FMN)和细胞色素f的拓扑结构， 一种与亚铁血红素共价连接的b-折叠蛋白。平衡生物物理 表征(圆二色谱、荧光、吸收、EXAFS、核磁共振和 不同的生化方法)将有助于揭示每个辅因子的作用 对其相应的蛋白质稳定性和未折叠的多肽结构进行分析。一个 用光化学方法启动折叠的最新技术 电子转移将被用来探测形成过程中的快速事件 蛋白质的天然状态。允许广泛的变性剂和时间范围 研究(和研究载脂蛋白)，时间分辨实验将 也可以使用停流混合进行。具体目标是：1. 表征辅因子配位和由 未折叠状态中的辅因子，2.研究铜、FMN和血红素如何 (与未折叠的多肽结合)影响多肽折叠动力学 最后，3.探索早期事件(从美国时间尺度开始) 形成--天然天青素、黄毒素和细胞色素。

DESCRIPTION: (Adapted from applicant's abstract) Cofactors may be important determinants, acting as nucleation points limiting the conformational search, for the folding rates of cofactor-binding proteins. To date, folding kinetics of proteins with b-sheet structure has not been as thoroughly investigated as for helical proteins. This research program aims towards probing the role of two inorganic and one organic cofactor in the folding of three proteins with mostly b-sheet structure. The targeted proteins are azurin, a b-barrel protein with a copper-ion cofactor, flavodoxin, a protein with an a/b doubly-wound topology coordinating an organic flavin mononucleotide (FMN), and cytochrome f, a b-sheet protein covalently linked to a heme. Equilibrium biophysical characterization (circular dichroism, fluorescence, absorption, EXAFS, NMR, and various biochemical methods) will aid in revealing the effect of each cofactor on its corresponding protein stability and unfolded polypeptide structure. A recent technique in which folding is initiated by photochemical electron-transfer will be used to probe rapid events during formation of the native-states of the proteins. To allow wide denaturant- and time- ranges to be investigated (and to study the apo proteins), time-resolved experiments will also be performed using stopped-flow mixing. The specific aims are to: 1. Characterize cofactor- coordination and residual structures created by the cofactors in the unfolded states, 2. Investigate how copper, FMN and heme (bound to the unfolded polypeptides) affect the polypeptide folding kinetics and, finally, 3. Probe early events (starting on the us time scale) during the formation -of native azurin, flavodoxin and cytochrome.